Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all oxygen evolving photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Industrial uses of carotenoids include pharmaceuticals, food supplements, electro-optic applications, animal feed additives, and colorants in cosmetics, to mention a few.
Because animals are unable to synthesize carotenoids de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved sources for carotenoids.
Carotenoids come in many different forms and chemical structures. Most naturally-occurring carotenoids are hydrophobic tetraterpenoids containing a C40 methyl-branched hydrocarbon backbone derived from successive condensation of eight C5 isoprene units (isopentenyl pyrophosphate, IPP). In addition, novel carotenoids with longer or shorter backbones occur in some species of nonphotosynthetic bacteria. The term “carotenoid” actually includes both carotenes and xanthophylls. A “carotene” refers to a hydrocarbon carotenoid. Carotene derivatives that contain one or more oxygen atoms, in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups, or within glycosides, glycoside esters, or sulfates, are collectively known as “xanthophylls”. Carotenoids are furthermore described as being acyclic, monocyclic, or bicyclic depending on whether the ends of the hydrocarbon backbones have been cyclized to yield aliphatic or cyclic ring structures (G. Armstrong, (1999) In Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321–352).
The genetics of carotenoid pigment biosynthesis are well known (Armstrong et al., J. Bact., 176: 4795–4802 (1994); Annu. Rev. Microbiol. 51:629–659 (1997)). This pathway is extremely well studied in the Gram-negative, pigmented bacteria of the genera Pantoea, formerly known as Erwinia. In both E. herbicola EHO-10 (ATCC 39368) and E. uredovora 20D3 (ATCC 19321), the crt genes are clustered in two operons, crt Z and crt EXYIB (U.S. Pat. No. 5,656,472, U.S. Pat. No. 5,545,816, U.S. Pat. No. 5,530,189, U.S. Pat. No. 5,530,188, and U.S. Pat. No. 5,429,939). Despite the similarity in operon structure, the DNA sequences of E. uredovora and E. herbicola crt genes show no homology by DNA-DNA hybridization (U.S. Pat. No. 5,429,939,).
The building block for carotenoids, IPP, is an isoprenoid. Isoprenoids constitute the largest class of natural products in nature, and serve as precursors for sterols (eukaryotic membrane stabilizers), gibberelinns and abscisic acid (plant hormones), menaquinone, plastoquinones, and ubiquinone (used as carriers for electron transport), as well as carotenoids and the phytol side chain of chlorophyll (pigments for photosynthesis). All isoprenoids are synthesized via a common metabolic precursor, isopentenyl pyrophosphate (IPP). Until recently, the biosynthesis of IPP was generally assumed to proceed exclusively from acetyl-CoA via the classical mevalonate pathway. However, the existence of an alternative mevalonate-independent pathway for IPP formation has been characterized for eubacteria and a green alga. E. coli contain genes that encode enzymes of the mevalonate-independent pathway of isoprenoid biosynthesis (FIG. 1). In this pathway, isoprenoid biosynthesis starts with the condensation of pyruvate with glyceraldehyde-3-phosphate (G3P) to form deoxy-D-xylulose via the enzyme encoded by the dxs gene. A host of additional enzymes are then used in subsequent sequential reactions, converting deoxy-D-xylulose to the final C5 isoprene product, isopentenyl pyrophosphate (IPP). IPP is converted to the isomer dimethylallyl pyrophophate (DMAPP) via the enzyme encoded by the idi gene. IPP is condensed with DMAPP to form C10 geranyl pyrophosphate (GPP) which is then elongated to C15 farnesyl pyrophosphate (FPP).
FPP synthesis is common in both carotenogenic and non-carotenogenic bacteria. E. coli do not normally contain the genes necessary for conversion of FPP to β-carotene (FIG. 1). Enzymes in the subsequent carotenoid pathway used to generate carotenoid pigments from FPP precursor can be divided into two categories: carotene backbone synthesis enzymes and subsequent modification enzymes. The backbone synthesis enzymes include geranyl geranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB), phytoene dehydrogenase (Crtl), and lycopene cyclase (CrtY/L), etc. The modification enzymes include ketolases, hydroxylases, dehydratases, glycosylases, etc.
Engineering E. coli for increased carotenoid production has previously focused on overexpression of key isoprenoid pathway genes from multi-copy plasmids. Various studies have report between a 1.5× and 50× increase in carotenoid formation in such E. coli systems upon cloning and transformation of plasmids encoding isopentenyl diphosphate isomerase (idi), geranylgeranyl pyrophosphate (GGPP) synthase (gps), deoxy-D-xylulose-5-phosphate (DXP) synthase (dxs), and DXP reductoisomerase (dxr) from various sources (Kim, S.-W., and Keasling, J. D., Biotech. Bioeng., 72:408–415 (2001); Mathews, P. D., and Wurtzel, E. T., Appl. Microbiol. Biotechnol., 53:396–400 (2000); Harker, M, and Bramley, P. M., FEBS Letter., 448:115–119 (1999); Misawa, N., and Shimada, H., J. Biotechnol., 59:169–181 (1998); Liao et al., Biotechnol. Bioeng., 62:235–241 (1999); Misawa et al., Biochem. J., 324:421–426 (1997); and Wang et al., Biotech. Bioeng., 62:235–241 (1999)).
Alternatively, other attempts to genetically engineer microbial hosts for increased production of carotenoids have focused on directed evolution of gps (Wang et al., Biotechnol. Prog., 16:922–926 (2000)) and overexpression of various isoprenoid and carotenoid biosynthetic genes in different microbial hosts using endogenous and exogenous promoters (Lagarde et al., Appl. Env. Microbiol., 66:64–72 (2000); Szkopinska et al., J. Lipid Res., 38:962–968 (1997); Shimada et al., Appl. Env. Microb., 64:2676–2680 (1998); and Yamano et al., Biosci. Biotech. Biochem., 58:1112–1114 (1994)).
Although these attempts at modulating carotenoid production have had some positive results, the production increases that can be effective by modulation of pathway enzymes is finite. For example, it has been noted that increasing isoprenoid precursor supply seems to be lethal (Sandmann, G., Trends in Plant Science, 6:14–17 (2001)), indicating limitations in the amount of carotenoid storage in E. coli. It is clear that alternate modifications will have to be made to achieve higher levels.
The problem to be solved therefore is to create a carotenoid overproducing organism for the production of new and useful carotenoids that do not involve direct manipulation of carotenoid or isoprenoid biosynthesis pathway genes. Applicants have solved the stated problem through the discovery that mutations in genes not involved in the isoprenoid or carotenoid biosynthetic pathways have a marked effect in increasing carotenoid production in a carotenoid producing microorganism.